1. Field of the Invention
This invention relates to technique for separating charged substances such as proteins, nucleic acids, cells, and the like, and more particularly to a free-flow electrophotoretic separation method and apparatus therefor which separates and extracts charged substances by utilizing electrophoresis.
2. Description of the Prior Art
An electrophoretic separation method, a membrane separation method and liquid chromatography are known conventionally as methods of separating and purifying charged substances such as proteins, nucleic acids, cells, and the like. The membrane separation method separates proteins by means of its pore size. Though capable of continuous processing, this method involves the problem that the separability of proteins is low. Liquid chromatography passes proteins to be separated through a carrier packed column and separates them. Though the separability is high, this method is not suitable for mass-processing on an industrial scale because the operation is carried out in batch-wise. The electrophoretic separation method separates and purifies proteins in an electric field by use of the difference of charge quantities of the proteins. This method can be classified into a carrier electrophoretic separation method using a carrier such as a gell, and a free-flow electrophoretic separation method which effects separation in a free flow without using a carrier. The carrier electrophoretic separation method is conducted batch-wise, and hence the free-flow electrophoretic separation method is suitable for mass-processing on an industrial scale.
The free-flow electrophoretic separation method is disclosed, for example, in Kurt Hannig's "Electrophoresis", 3, p.p. 235-243, 1982, West Germany. This method will be described in further detail.
A mixture of proteins to be separated is charged continuously into a separation buffer that flow down at a constant speed across an electric field inside an electrophoretic chamber. Since each protein has a different charge quantity, its electrophoretic mobility is also different in the electric field. Therefore, each protein is deflected and separated while it flows down in the separation buffer in conjunction with the flowing velocity of the separation buffer. It can thus be understood that this method can continuously separate the proteins and hence is effective for the separation and purification of the proteins on an industrial scale.
In order to improve the separability by this method, it is important to always keep constant the flowing velocity of the separation buffer inside the separation chamber. However, since a current is caused to flow through the separation buffer, joule heat occurs necessarily, and this heat causes convection in the separation buffer and hence, turbulence of the flow of the separation buffer. In consequence, the separability of the proteins drops. To obviate this problem, it has been necessary for a conventional free-flow electrophoretic separation apparatus to control the temperature and flowing velocity of the separation buffer to a level of accuracy as high as .+-.0.2%. To accomplish this object, the separation chamber must be miniaturized in a thin flat sheet form, and this in turn results in the practical problem that the processing quantity is too small to conduct the separation on a large industrial scale.
U.S. Pat. No. 3,989,613 discloses an electrophoretic separation apparatus having a construction which comprises two electrophoretic chambers divided by a boundary membrane consisting of a semipermeable membrane, electrodes and electrode chambers disposed on both sides of the boundary membrane, cooling chambers each disposed between each electrode chamber and each electrophoretic chamber, and liquid inlets and outlets disposed at the upper and lower portions of each of the two electrophoretic chambers. The separation of the charged substances is effected by this apparatus in the following manner. A separation buffer is supplied from the upper inlet of one of the two electrophoretic chambers, while a separation buffer which contains the two charged substances to be separated are mixed is supplied from the upper inlet of the other electrophoretic chamber. A d.c. voltage is then applied across both electrodes for separation. The separation buffer and the solution of the charged substances flow down inside the electrophoretic chambers, and some of the charged substances permeate through the boundary membrane at the center into the other electrophoretic chamber from the electrophoretic chamber on the supply side due to electrophoresis, while the other flows down through the electrophoretic chamber on the supply side. The two charged substances are then withdrawn from the lower outlets of the respective electrophoretic chambers. In this apparatus, too, the thickness of each electrophoretic chamber is extremely small in order to eliminate any adverse influences of the joule heat caused by the application of the d.c. voltage. For this reason, the processing quantity is small and the apparatus can not be adapted to mass processing.